Murine RATH gene

ABSTRACT

The present invention relates, first, to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules, or degenerate variants thereof, that participate in the regulation, control and/or modulation of G-protein-mediated signal transduction involved in T cell activation, including, but not limited to T helper (TH) cell and TH cell subpopulation activation. Specifically, the nucleic acid molecules of the present invention include the genes corresponding to the mammalian R11 gene, including the human R11 gene. R11 sequence analysis indicates that the R11 gene is a novel gene belonging to the RGS (&#34;regulator of G-protein signalling&#34;) gene family, a gene family which encodes gene products involved in G-protein-mediated signal transduction.

1. INTRODUCTION

The present invention relates, first, to the identification of novel nucleic acid molecules and polypeptides encoded by such nucleic acid molecules, or degenerate variants thereof, that participate in the modulation of T cell activity, including, but not limited to T helper (TH) cell and TH cell subpopulation activity. Such nucleic acids and polypeptides are important, for example, in developing diagnostics and targets for intervention therapy. Specifically, the nucleic acid molecules of the present invention include the genes corresponding to the mammalian RATH genes, including the RATH1.1 gene. Sequence analysis indicates that the RATH gene is a novel gene belonging to the RGS ("regulator of G-protein signalling") gene family, a gene family which encodes gene products involved in G-protein-mediated signal transduction that act to inhibit G-protein-mediated signalling at the level of receptor/G-protein interaction or the G protein α subunit itself. The discovery of the RATH1.1 gene represents the first identification of a T cell specific--and more particularly, a T helper (TH) cell subpopulation specific--RGS gene and gene product. The present invention further relates to methods for the diagnostic evaluation, prognosis and treatment of immune disorders involving T cell activation, including, but not limited to, TH cell and TH cell subpopulation activations. Such immune disorders include, but are not limited to chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Gravels disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy. The present invention still further relates to methods of treating the above diseases by administering compounds which modulate RATH proteins and/or RATH gene activity.

2. BACKGROUND OF THE INVENTION

Two distinct types of T lymphocytes are recognized: CD8⁺ cytotoxic T lymphocytes (CTLS) and CD4⁺ helper T lymphocytes (TH cells). CTLs recognize and kill cells which display foreign antigens of their surfaces. CTL precursors display T cell receptors that recognize processed peptides derived from foreign proteins, in conjunction with class I MHC molecules, on other cell surfaces. This recognition process triggers the activation, maturation and proliferation of the precursor CTLs, resulting in CTL clones capable of destroying the cells exhibiting the antigens recognized as foreign.

Human and murine TH cells are involved in both humoral and cell-mediated forms of effector immune responses. With respect to the humoral, or antibody, immune response, antibodies are produced by B lymphocytes through interactions with TH cells. In murine cells, extracellular antigens are endocytosed by antigen-presenting cells (APCs), processed, and presented preferentially in association with class II major histocompatibility complex (MHC) molecules to CD4⁺ class II MHC-restricted TH cells. These TH cells in turn activate B lymphocytes, resulting in antibody production.

The cell-mediated, or cellular, immune response, functions to neutralize microbes which inhabit intracellular locations. Foreign antigens, such as, for example, viral antigens, are synthesized within infected cells and presented on the surfaces of such cells in association with class I MHC molecules. This, then, leads to the stimulation of the CD8⁺ class I MHC-restricted CTLs.

Some agents, such as mycobacteria, which cause tuberculosis and leprosy, are engulfed by macrophages and processed in vacuoles containing proteolytic enzymes and other toxic substances. While these macrophage components are capable of killing and digesting most microbes, agents such as mycobacteria survive and multiply. The agents' antigens are processed, though, by the macrophages and presented preferentially in association with class II MHC molecules to CD4⁺ class II MHC-restricted TH cells, which become stimulated to secrete interferon-γ, which, in turn, activates macrophages. As a result of such activation, the cells exhibit increased bacteriocidal ability.

Murine TH cells are composed of at least two distinct subpopulations, termed TH1 and TH2 cell subpopulations. Evidence suggests that TH1 and TH2 subtypes represent extremely polarized populations of TH cells. While such subpopulations were originally discovered in murine systems (reviewed in Mosmann, T. R. and Coffman, R. L., 1989, Ann. Rev. Immunol. 7:145), the existence of TH1- and TH2-like subpopulations has also been established in humans (Del Prete, A. F. et al., 1991, J. Clin. Invest. 88:346; Wiernenga, E. A. et al., 1990, J. Imm. 144:4651; Yamamura, M. et al., 1991, Science 254:277; Robinson, D. et al., 1993, J. Allergy Clin. Imm. 92:313). While TH1-like and TH2-like cells can represent the most extremely polarized TH cell subpopulations, other TH cell subpopulations, such as TH0 cells (Firestein, G. S. et al., 1989, J. Imm. 143:518), which represent TH cells which have characteristics of TH1 and TH2 cell subpopulations.

TH1-like and TH2-like cells appear to function as part of the different effector functions of the immune system (Mosmann, T. R. and Coffmann, R. L., 1989, Ann. Rev. Imm. 7:145). Specifically, TH1-like cells direct the development of cell-mediated immunity, triggering phagocyte-mediated host defenses, and are associated with delayed hypersensitivity. Accordingly, infections with intracellular microbes tend to induce TH1-type responses. TH2 cells drive humoral immune responses, which are associated with, for example, defenses against certain helminthic parasites, and are involved in antibody and allergic responses.

It has been noted that the ability of the different TH cell types to drive different immune effector responses is due to the exclusive combinations of cytokines which are expressed within a particular TH cell subpopulation. For example, TH1 cells are known to secrete interleukin-2 (IL-2), interferon-γ (IFN-γ), and lymphotoxin, while TH2 cells secrete interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-10 (IL-10).

It has recently been noted that, in addition to CD4⁺ TH cells, CD8⁺ CTLs can, under certain conditions, also exhibit TH1-like or TH2-like cytokine profiles (Seder, R. A. et al., 1995, J. Exp. Med. 181:5-7; Manetti, R. et al., 1994, J. Exp. Med. 180:2407-2411; Maggi, E. et al., 1994, J. Exp. Med. 180:489-495). While the precise functional role of such CD8⁺ TH-like cells is currently unknown, these cell subpopulations appear to have great relevance to immune responses against infectious agents such as viruses and intracellular parasites.

Failure to control or resolve an infectious process often results from an inappropriate immune response. Such an inappropriate immunological response can include, for example, atopic conditions (i.e., IgE-mediated allergic conditions) such as asthma, allergy, including allergic rhinitis, dermatitis, including psoriasis, pathogen susceptibilities, chronic inflammatory disease, organ-specific autoimmunity, graft rejection and graft versus host disease. Failure to resolve infestation by helminthic parasites is an example of a condition which may result in an inappropriate immune response.

Further, while TH1-mediated inflammatory responses to many pathogenic microorganisms are beneficial, such responses to self antigens are usually deleterious. It has been suggested that the preferential activation of TH1-like responses is central to the pathogenesis of such human inflammatory autoimmune diseases as multiple sclerosis and insulin-dependent diabetes. For example, TH1-type cytokines predominate in the cerebrospinal fluid of patients with multiple sclerosis, pancreases of insulin-dependent diabetes patients, thyroid glands of Hashimoto's thyroiditis, and gut of Crohn's disease patients, suggesting that such patients mount a TH1-like, not a TH2-like, response to the antigen(s) involved in the etiopathogenesis of such disorders.

Accordingly, it has become increasingly important to identify T cell specific genes, gene products, in particular, TH cell and TH cell subpopulation specific genes and gene products, for use as targets for drug intervention therapy.

3. SUMMARY OF THE INVENTION

The present invention relates, first, to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules, or degenerate variants thereof, that participate in the modulation of G-protein-mediated signal transduction involved in T cell activity, including, but not limited to T helper (TH) cell and TH cell subpopulation activity. Specifically, the nucleic acid molecules of the present invention include the genes corresponding to the mammalian RATH genes, including the RATH1.1 gene.

In particular, the compositions of the present invention include nucleic acid molecules (e.g., RATH genes), including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants, which encode novel RATH gene products, and antibodies directed against such RATH gene products or conserved variants or fragments thereof. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which have been transformed with such nucleic acid molecules.

The nucleic acid sequence of the murine RATH1.1 (also referred to as femtR11) gene is provided. A 2278 bp RATH1.1 cDNA was deposited on Jul. 30, 1996 with the American Type Culture Collection (ATCC), bearing ATCC Accession No. 98116. The RATH1.1 gene produces a transcript of approximately 3 kb and encodes a protein of 201 amino acids with a molecular weight of approximately 22.6 kD, the sequence of which is provided. Transcripts were detected in stimulated TH cell subpopulations, in a TH1-specific fashion. The amino acid sequence of the predicted full length RATH1.1 gene product does not contain either a recognizable transmembrane domain or a signal sequence, suggesting that the RATH1.1 gene product is an intracellular or membrane-associated gene product. Sequence analysis indicates that the RATH1.1 gene is a novel gene having an RGS ("regulator of G-protein signalling") motif at approximately amino acid residue 60 to approximately amino acid residue 179, as depicted in FIGS. 1A-1C. Thus, RATH1.1 belongs to a gene family, termed the RATH gene family, which encodes RGS-containing gene products involved in G-protein-mediated signal transduction that act to inhibit G-protein-mediated signalling at the level of receptor/G-protein interaction or the G protein α subunit itself. The RATH1.1 gene and the RATH gene family represents the first identification of T cell specific--and more particularly, T helper (TH) cell subpopulation specific--RGS genes and gene products.

The mammalian RATH gene is, as shown herein, expressed in a highly regulated and restricted manner. Specifically, RATH1.1 is expressed in a highly TH1-inducible manner. That is, the RATH1.1 gene is highly upregulated in stimulated TH1 cell subpopulations and appears to be absent from, or present in very low levels within, other cell types.

The present invention further relates to methods for the diagnostic evaluation and prognosis of immune disorders involving T cell activation, including, but not limited to, TH cell and TH cell subpopulation activation. Such immune disorders include, but are not limited to chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (eag., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy. The present invention also relates to methods for the identification of subjects having a predisposition to such conditions. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of RATH gene mutations, allelic variations and regulatory defects in the RATH gene.

Further, methods and compositions are presented for the treatment of immune disorders involving T cell activation, including, but not limited to, TH cell and TH cell subpopulation activation, such as those listed above. Such methods and compositions are capable of modulating the level of RATH gene expression and/or the level of RATH gene product activity.

Still further, the present invention relates to methods for the use of the RATH gene and/or RATH gene products for the identification of compounds which modulate RATH gene expression and/or the activity of RATH gene products. Such compounds can be used as agents to control, prevent, treat and/or ameliorate (hereinafter referred to as "control") the severity of immune disorders such as those listed above.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1C RATH1.1 nucleotide and amino acid sequence (SEQ ID NO:2). Top line: RATH1.1 amino acid sequence; bottom line: RATH1.1 nucleotide sequence (SEQ ID NO:1).

5. DETAILED DESCRIPTION OF THE INVENTION

Described herein are the identification of the novel mammalian RATH (RATH) genes, including the RATH1.1 gene, which are involved in the control of immune disorders by modulating T cell activity, including, but not limited to, TH cell and TH cell subpopulation activity. Such immune disorders include, but are not limited to chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.

Specifically described are recombinant mammalian RATH DNA molecules, cloned genes, or degenerate variants thereof. The compositions of the present invention further include RATH gene products (e.g., proteins) that are encoded by the RATH gene. Also described herein are antibodies against RATH gene products (e.g., proteins), or conserved variants or fragments thereof, and nucleic acid probes useful for the identification of RATH gene mutations and the use of such nucleic acid probes in diagnosing immune disorders involving T cell activation, including TH cell and TH cell subpopulation activation, such as those listed above.

Further described are methods for the use of the RATH gene and/or RATH gene products in the identification of compounds which modulate the activity of the RATH gene product. Methods are also described for the treatment of immune disorders involving T cell activation, including TH cell and TH cell subpopulation activation, via modulation of RATH1.1 activity including, for example, modulation of gene expression and the ability to bind a G protein coupling receptor.

The RATH1.1 nucleic acid compositions of the invention are described in the Example presented, below, in Section 6.

5.1. THE RATH GENE

The RATH1.1 gene, shown in FIGS. 1A-1C (SEQ ID NOS: 1 and 2), is a novel RGS gene which is regulated in a TH manner, specifically, in a TH1inducible manner. RATH1.1 is an exemplary RATH gene and represents the first example of a T cell specific RGS-containing gene, more particularly a TH cell subpopulation specific gene. As such, RATH genes can be involved in T cell activity, including TH cell and TH cell subpopulation activation.

Nucleic acid sequences of the identified RATH gene are described herein. As used herein, "RATH gene" refers to (a) a gene containing the DNA sequence shown in FIGS. 1A-1C (SEQ ID NO:1), or contained in the cDNA clone within E. coli femtR11A as deposited with the American Type Culture Collection (ATCC) on Jul. 30, 1996, bearing ATCC Accession No. 98116; (b) any DNA sequence that encodes the amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO:2), or encoded by the cDNA clone within E. coli femtR11A, as deposited with the ATCC (ATCC Accession No. 98116) on Jul. 30, 1996; (c) any DNA sequence that hybridizes to the complement of the DNA sequences that encode the amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO:2), or contained in the cDNA clone within E. coli femtR11A as deposited with the ATCC (ATCC Accession No. 98116), under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) or (d) any DNA sequence that hybridizes to the complement of the DNA sequences that encode the amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO:2), or contained in the cDNA clone within E. coli femtR11A as deposited with the ATCC (ATCC Accession No. 98116), under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), and encodes a gene product functionally equivalent to an RATH gene product encoded by sequences contained in the cDNA clone within E. coli femtR11A, or sequences shown in FIGS. 1A-1C (SEQ ID NO: 1), yet which still encodes a functionally equivalent RATH gene product.

As used herein, RATH gene may also refer to fragments and/or degenerate variants of DNA sequences (a) through (d), especially naturally occurring variants thereof. A preferred RATH gene fragment comprises a nucleic acid molecule which encodes the RGS domain contained within the amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO:2).

A RATH gene sequence preferably exhibits at least about 80% overall similarity at the nucleotide level to the nucleic acid sequence depicted in FIGS. 1A-1C (SEQ ID NO:1), more preferably exhibits at least about 85-90% overall similarity to the FIGS. 1A-1C nucleic acid sequence (SEQ ID NO:1) and most preferably exhibits at least about 95% overall similarity to the FIGS. 1A-1C (SEQ ID NO:1), nucleic acid sequence.

Preferably, the RATH gene is expressed in a TH specific or enhanced manner, and more preferably, the RATH gene is expressed in a TH cell subpopulation specific or enhanced manner.

The RATH gene sequences of the invention further include isolated nucleic acid molecules which hybridize under highly stringent or moderate stringent conditions to at least about 6, preferably about 12, more preferably about 18, consecutive nucleotides of the RATH gene sequences of (a)-(d).

The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences (a) through (d), in the preceding paragraph. Such hybridization conditions may be highly stringent or moderately stringent, as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e.g., to washing in 6×SSC/0.050 sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). These nucleic acid molecules may encode or act as RATH gene antisense molecules useful, for example, in RATH gene regulation (for example, as antisense primers in amplification reactions of RATH gene nucleic acid sequences. With respect to RATH gene regulation, such techniques can be used to modulate, for example, T cell activation via activation or inhibition of G-protein-mediated signal transduction.

Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for RATH gene regulation. Still further, such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular RATH allele or alternatively spliced RATH transcript responsible for causing or predisposing one to an immune disorder involving T cell activation may be detected.

Still further, the invention encompassing RATH genes as a screen in an engineered yeast system, including, but not limited to, the yeast two hybrid system described herein.

The invention also encompasses (a) DNA vectors that contain any of the foregoing RATH coding sequences and/or their complements (e.g., antisense); (b) DNA expression vectors that contain any of the foregoing RATH coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing RATH coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include, but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors.

The invention includes fragments of any of the DNA sequences disclosed herein.

In addition to the RATH gene sequences described above, homologs of such sequences, exhibiting extensive homology to one or more of domains of the RATH gene product present in other species can be identified and readily isolated, without undue experimentation, by molecular biological techniques well known in the art. Further, there can exist homolog genes at other genetic loci within the genome that encode proteins which have extensive homology to one or more domains of the RATH gene product. These genes can also be identified via similar techniques. Still further, there can exist alternatively spliced variants of the RATH1.1 gene.

As an example, in order to clone a human RATH gene homolog or variants using isolated murine RATH gene sequences as disclosed herein, such murine RATH gene sequences are labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues (e.g., peripheral lymphocytes, activated T cells, preferably activated TH cells) derived from the organism (in this case, human) of interest. With respect to the cloning of such a human RATH homolog, a human activated T cell cDNA library may, for example, be used for screening. In particular, a cDNA library made from human peripheral lymphocytes which are stimulated with phytohemagglutinin (PHA) for 48 hours.

The hybridization and wash conditions used should be of a low stringency when the cDNA library is derived from a different type of organism than the one from which the labeled sequence was derived. Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

With respect to the cloning of a human RATH homolog, using murine RATH1.1 sequences, for example, various stringency conditions which promote DNA hybridization can be used. For example, hybridization in 6×SSC at about 45° C., followed by washing in 2×SSC at 50° C. may be used. Alternatively, the salt concentration in the wash step can range from low stringency of about 5×SSC at 50° C., to moderate stringency of about 2×SSC at 50° C., to high stringency of about 0.2×SSC at 50° C. In addition, the temperature of the wash step can be increased from low stringency conditions at room temperature, to moderately stringent conditions at about 42° C., to high stringency conditions at about 65° C. Other conditions include, but are not limited to, hybridizing at 68° C. in 0.5M NaHPO₄ (pH7.2)/ 1 mM EDTA/ 7% SDS, or hybridization in 50% formamide/0.25M NaHPO₄ (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; followed by washing in 40 mM NaHPO₄ (pH 7.2)/l mM EDTA/5% SDS at 50° C. or in 40 mM NaHPO₄ (pH7.2) 1 mM EDTA/1% SDS at 50° C. Both temperature and salt may be varied, or alternatively, one or the other variable may remain constant while the other is changed.

Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions well known to those of skill in the art.

Further, a RATH gene homolog may be isolated from nucleic acid of the organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the RATH gene roduct disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express a RATH gene allele.

The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a RATH gene nucleic acid sequence. The PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

PCR technology may be utilized to isolate full length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (e.g., one known, or suspected, to express the RATH gene, such as, for example, lymphocytes and activated T cells, especially activated TH cells). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies which may be used, see e.g., Sambrook et al., 1989, supra.

RATH gene sequences may additionally be used to isolate mutant RATH gene alleles. Such mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of immune disorders involving T cell activity, including TH cell and TH cell subpopulation activity, such as those listed above. Mutant alleles and mutant allele products may then be utilized in the screening, therapeutic and diagnostic systems described herein. Additionally, such RATH gene sequences can be used to detect RATH gene regulatory (e.g., promoter) defects which can affect such immune disorders.

A cDNA of a mutant RATH gene may be isolated, for example, by using PCR, a technique which is well known to those of skill in the art. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant RATH allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant RATH allele to that of the normal RATH allele, the mutation(s) responsible for the loss or alteration of function of the mutant RATH gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry the mutant RATH allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express the mutant RATH allele. The normal RATH gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant RATH allele in such libraries. Clones containing the mutant RATH gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant RATH allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal RATH gene product, as described, below, in Section 5.3. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Anti-bodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) In cases where a RATH mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of anti-RATH gene product antibodies are likely to cross-react with the mutant RATH gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

5.2. PROTEIN PRODUCTS OF THE RATH GENE

RATH gene products, or peptide fragments thereof, can be prepared for a variety of uses. For example, such gene products, or peptide fragments thereof, can be used for the generation of antibodies, in diagnostic assays, or for the identification of other cellular gene products involved in the modulation of T cell activity, such as, for example, T cell activation, including TH cell and TH cell subpopulation activity, such as, for example, TH cell and TH cell subpopulation activation.

The amino acid sequence depicted in FIGS. 1A-1C represents a RATH gene product. The RATH gene product, sometimes referred to herein as a "RATH protein", may additionally include those gene products encoded by the RATH gene sequences described in Section 5.1, above.

In addition, RATH gene products may include proteins that represent functionally equivalent gene products. Such an equivalent RATH gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the RATH gene sequences described, above, in Section 5.1, but which result in a silent change, thus producing a functionally equivalent RATH gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In a particular embodiment, a RATH gene product comprises the RGS domain contained within the amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO:2). The RGS domain lies from about amino acid residue 60 to about amino acid residue 179, as depicted in FIGS. 1A-1C (SEQ ID NO:2).

"Functionally equivalent", as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous RATH gene products encoded by the RATH gene sequences described in Section 5.1, above. The in vivo activity of the RATH gene product, as used herein, refers to the ability of the RATH gene product, when present in an appropriate cell type, to desensitize T cell-relevant physiological stimuli, such as may, for example, occur in allergic inflammation disease or autoimmune disease.

In addition, "functionally equivalent", refers to a protein capable of exhibiting substantially similar in vivo activity in yeast as the SST2 gene product which is the yeast homolog of RATH. Mutations in SST2 cause increased sensitivity and defective desensitization to the yeast α factor mating pheromone. α-factor binds to a G-protein coupled receptor and causes cells to arrest in the G1 phase of the cell cycle. A zone of inhibition assay measures pheromone sensitivity and desensitization (Dohlman et al., 1995, Mol. Cell Biol., 15:3635-3643). In this assay filter disks impregnated with alpha factor were placed on top of a lawn of cells of a MATa yeast strain to be tested. The alpha factor diffuses away form the disk into the agar creating a gradient of alpha factor concentration. Relative to wild-type cells, SST2 mutants have larger zones of growth inhibition and the cells within these halos do not desensitize to the pheromone signal by resuming growth. A "functionally equivalent" RATH would refer to a protein able to compliment the yeast SST2 mutation, including, but not limited to the ability to interact with substantially similar polypeptides in a yeast two hybrid system described herein.

A RATH gene product sequence preferably exhibits at least about 80% overall similarity at the amino acid level to the amino acid sequence depicted in FIGS. 1A-1C (SEQ ID NO:2), more preferably exhibits at least about 90% overall similarity to the FIGS. 1A-1C amino acid sequence and most preferably exhibits at least about 95% overall similarity to the FIGS. 1A-1C amino acid sequence (SEQ ID NO:2).

RATH gene products can also include fusion proteins comprising a RATH gene product sequence as described in this section operatively associated to a heterologous, e.g., peptide, component. Heterologous components can include, but are not limited to sequences which faciliate isolation and purification of fusion protein, or label components. Such isolation and label components are well known to those of skill in the art.

The RATH gene products or peptide fragments thereof, may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the RATH gene polypeptides and peptides of the invention by expressing nucleic acid containing RATH gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing RATH gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra. Alternatively, RNA capable of encoding RATH gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to express the RATH gene coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the RATH gene product of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing RATH gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the RATH gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the RATH gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing RATH gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the RATH gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of RATH protein or for raising antibodies to RATH protein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the RATH gene product coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiderda cells. The RATH gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of RATH gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g., see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the RATH gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing RATH gene product in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted RATH gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire RATH gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the RATH gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, T cell lines such as, for example, Jurkat, CTLL, HT2, Dorris, D1.1, AE7, D10.G4 and CDC25.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the RATH gene product may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the RATH gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the RATH gene product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺. nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

The RATH gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate RATH transgenic animals.

Any technique known in the art may be used to introduce the RATH gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the RATH transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the RATH gene transgene be integrated into the chromosomal site of the endogenous RATH gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous RATH gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous RATH gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous RATH gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu, et al., 1994, Science 265: 103-106). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Methods for the production of single-copy transgenic animals with chosen sites of integration are also well known to those of skill in the art. See, for example, Bronson et al. (Bronson, S. K. et al., 1996, Proc. Natl. Acad. Sci. USA 93:9067-9072), which is incorporated herein by reference in its entirety.

Once transgenic animals have been generated, the expression of the recombinant RATH gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of RATH gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the RATH transgene product.

5.3. ANTIBODIES TO RATH GENE PRODUCTS

Described herein are methods for the production of antibodies capable of specifically recognizing one or more RATH gene product epitopes or epitopes of conserved variants or peptide fragments of the RATH gene products.

Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a RATH gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of RATH gene products, and/or for the presence of abnormal forms of the such gene products. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, in Section 5.4.2, for the evaluation of the effect of test compounds on RATH gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, in Section 5.4.3, to, for example, evaluate the normal and/or engineered RATH-expressing cells prior to their introduction into the patient.

Anti-RATH gene product antibodies may additionally be used as a method for the inhibition of abnormal RATH gene product activity. Thus, such antibodies may, therefore, be utilized as part of immune disorder treatment methods.

For the production of antibodies against a RATH gene product, various host animals may be immunized by injection with a RATH gene product, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium Parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a RATH gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with RATH gene product supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce single chain antibodies against RATH gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

5.4. USES OF THE RATH GENE, GENE PRODUCTS, AND ANTIBODIES

Described herein are various applications of the RATH gene, the RATH gene product including peptide fragments thereof, and of antibodies directed against the RATH gene product and peptide fragments thereof. Such applications include, for example, prognostic and diagnostic evaluation of immune disorders involving T cell activation, including TH cell and TH cell subpopulation activation, and the identification of subjects with a predisposition to such disorders, as described, below, in Section 5.4.1. Additionally, such applications include methods for the treatment of such immune disorders, as described, below, in Section 5.4.2, and for the identification of compounds which modulate the expression of the RATH gene and/or the activity of the RATH gene product, as described below, in Section 5.4.3. Such compounds can include, for example, other cellular products which are involved in T cell activation, especially processes relating to G-protein-mediated signal transduction in T cells. These compounds can be used, for example, in the amelioration of immune disorders involving T cell activation, including TH cell and TH cell subpopulation activation.

5.4.1. DIAGNOSIS OF IMMUNE DISORDER ABNORMALITIES

A variety of methods can be employed for the diagnostic and prognostic evaluation of immune disorders involving T cell activation, including, but not limited to, TH cell and TH cell subpopulation activity, such as T cell activation. Such immune disorders include, but are not limited to chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy. Methods are further provided for the identification of subjects having a predisposition to such disorders.

Such methods may, for example, utilize reagents such as the RATH gene nucleotide sequences described in Sections 5.1, and antibodies directed against RATH gene products, including peptide fragments thereof, as described, above, in Section 5.2. Specifically, such reagents may be used, for example, for: (1) the detection of the presence of RATH gene mutations, or the detection of either over- or under-expression of RATH gene mRNA relative to the non-immune disorder state or the qualitative or quantitative detection o f alternatively spliced forms of RATH transcripts which may correlate with certain immune disorders or susceptibility toward such body disorders; and (2) the detection of either an over- or an under-abundance of RATH gene product relative to the non-immune disorder state or the presence of a modified (e.g., less than full length) RATH gene product which correlates with an immune disorder state or a progression toward an immune disorder state.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic test kits comprising at least one specific RATH gene nucleic acid or anti-RATH gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to screen and diagnose patients exhibiting immune disorder abnormalities and to screen and identify those individuals exhibiting a predisposition to such an immune disorder abnormality.

For the detection of RATH mutations, any nucleated cell can be used as a starting source for genomic nucleic acid. For the detection of PATH transcripts or RATH gene products, any cell type or tissue in which the RATH gene is expressed, such as, for example, activated T cells, including activated TH cells, may be utilized.

Nucleic acid-based detection techniques are described, below, in Section 5.4.1.1. Peptide detection techniques are described, below, in Section 5.4.1.2.

5.4.1.1 DETECTION OF RATH GENE NUCLEIC ACID MOLECULES

Mutations or polymorphisms within the RATH gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures which are well known to those of skill in the art.

Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving RATH gene structure, including point mutations, insertions, deletions and chromosomal rearrangements. Such assays may include, but are not limited to, direct sequencing (Wong, C. et al., 1987, Nature 330:384-386), single stranded conformational polymorphism analyses (SSCP; Orita, M. et al., 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), heteroduplex analysis (Keen, T. J. et al., 1991, Genomics 11:199-205; Perry, D. J. & Carrell, R. W., 1992), denaturing gradient gel electrophoresis (DGGE; Myers, R. M. et al., 1985, Nucl. Acids Res. 13:3131-3145), chemical mismatch cleavage (Cotton, R. G. et al., 1988, Proc. Natl. Acad. Sci. USA 85:4397-4401) and oligonucleotide hybridization (Wallace, R. B. et al., 1981, Nucl. Acids Res. 9:879-894; Lipshutz, R. J. et al., 1995, Biotechniques 19:442-447).

Diagnostic methods for the detection of RATH gene specific nucleic acid molecules, in patient samples or other appropriate cell sources, may involve the amplification of specific gene sequences, e.g., by the polymerase chain reaction (PCR; the experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art, such as, for example, those listed above. Utilizing analysis techniques such as these, the amplified sequences can be compared to those which would be expected if the nucleic acid being amplified contained only normal copies of the RATH gene in order to determine whether a RATH gene mutation exists.

Further, well-known genotyping techniques can be performed to type polymorphisms that are in close proximity to mutations in the RATH gene itself. These polymorphisms can be used to identify individuals in families likely to carry mutations. If a polymorphism exhibits linkage disequilibrium with mutations in the RATH gene, it can also be used to identify individuals in the general population likely to carry mutations. Polymorphisms that can be used in this way include restriction fragment length polymorphisms (RFLPs), which involve sequence variations in restriction enzyme target sequences, single-base polymorphisms and simple sequence repeat polymorphisms (SSLPs).

For example, Weber (U.S. Pat. No. 5,075,217, which is incorporated herein by reference in its entirety) describes a DNA marker based on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n short tandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks is estimated to be 30,000-60,000 bp. Markers which are so closely spaced exhibit a high frequency co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the RATH gene, and the diagnosis of diseases and disorders related to RATH mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is incorporated herein by reference in its entirety) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the RATH gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.

A RATH probe could additionally be used to directly identify RFLPs. Additionally, a RATH probe or primers derived from the RATH sequence could be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids. The DNA contained in these clones can be screened for single-base polymorphisms or simple sequence length polymorphisms (SSLPs) using standard hybridization or sequencing procedures.

Alternative diagnostic methods for the detection of RATH gene-specific mutations or polymorphisms can include hybridization techniques which involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the RATH gene. Preferably, the lengths of these nucleic acid reagents are at least to 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid:RATH molecule hybrid. The presence of nucleic acids which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled RATH nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The RATH gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal RATH gene sequence in order to determine whether a RATH gene mutation is present.

Quantitative and qualitative aspects of RATH gene expression can also be assayed. For example, RNA from a cell type or tissue known, or suspected, to express the RATH gene, such as activated T cells, including activated TH cells, may be isolated and tested utilizing hybridization or PCR techniques such as are described, above. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the RATH gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the RATH gene, including activation or inactivation of RATH gene expression and presence of alternatively spliced RATH transcripts.

In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). All or part of the resulting cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the RATH gene nucleic acid reagents described in Section 5.1. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides.

For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.

Such RT-PCR techniques can be utilized to detect differences in RATH transcript size which may be due to normal or abnormal alternative splicing. Additionally, such techniques can be performed using standard techniques to detect quantitative differences between levels of full length and/or alternatively spliced RATH transcripts detected in normal individuals relative to those individuals exhibiting immune disorders or exhibiting a predisposition to toward such immune disorders.

In the case where detection of specific alternatively spliced species is desired, appropriate primers and/or hybridization probes can be used, such that, in the absence of such sequence, no amplification would occur. Alternatively, primer pairs may be chosen utilizing the sequence data depicted in FIGS. 1A-1C (SEQ ID NO:1), to choose primers which will yield fragments of differing size depending on whether a particular exon is present or absent from the transcript RATH transcript being utilized.

As an alternative to amplification techniques, standard Northern analyses can be performed if a sufficient quantity of the appropriate cells can be obtained. Utilizing such techniques, quantitative as well as size related differences between RATH transcripts can also be detected.

Additionally, it is possible to perform such RATH gene expression assays "in situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, N.Y.).

5.4.1.2. DETECTION OF RATH GENE PRODUCTS

Antibodies directed against wild type or mutant RATH gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.2, may also be used as immune disorder diagnostics and prognostics, as described herein. Such diagnostic methods, may be used to detect abnormalities in the level of RATH gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of RATH gene product. Because evidence disclosed herein indicates that the RATH gene product is an intracellular gene product, the antibodies and immunoassay methods described below have important in vitro applications in assessing the efficacy of treatments for immune disorders such as those described herein. Antibodies, or fragments of antibodies, such as those described below, may be used to screen potentially therapeutic compounds in vitro to determine their effects on RATH gene expression and RATH peptide production. The compounds which have beneficial effects on immune disorders can be identified and a therapeutically effective dose determined.

In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for immune disorders involving T cell activation, including TH cell and TH cell subpopulation activation. Antibodies directed against RATH peptides may be used in vitro to determine the level of RATH gene expression achieved in cells genetically engineered to produce RATH peptides. Given that evidence disclosed herein indicates that the RATH gene product is an intracellular gene product, such an assessment is, preferably, done using cell lysates, membrane associated fractions or extracts. Such analysis will allow for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.

The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the RATH gene, such as, for example, activated T cells. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cell taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the RATH gene.

Preferred diagnostic methods for the detection of RATH gene products or conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the RATH gene products or conserved variants, including gene products which are the result of alternatively spliced transcripts, or peptide fragments are detected by their interaction with an anti-RATH gene product-specific antibody.

For example, antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, useful in the present invention may be used to quantitatively or qualitatively detect the presence of RATH gene products or conserved variants or peptide fragments thereof. The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of RATH gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the RATH gene product, or conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays for RATH gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying RATH gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled RATH gene specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound, antibody. The amount of bound label on solid support may then be detected by conventional means.

By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-RATH gene product antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

One of the ways in which the RATH gene peptide-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect RATH gene peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the hemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

5.4.2. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE RATH ACTIVITY

RATH is involved in the control of immune disorders involving T-cell activity. At least one specific RATH activity is its role in T-helper cell activation.

The following assays are designed to identify: (i) compounds that bind to RATH gene products; (ii) compounds that bind to other intracellular proteins that interact with a RATH gene product; (iii) compounds that interfere with the interaction of the RATH gene product with other intracellular proteins; and (iv) compounds that modulate the activity of RATH gene (i.e., modulate the level of RATH gene expression and/or modulate the level of RATH gene product activity). In a preferred embodiment of such assays, are assays which identify compounds that alter RATH binding to intracellular G-α subunits.

Assays may additionally be utilized which identify compounds which bind to RATH gene regulatory sequences (e.g., promoter sequences). See e.g., Platt, K. A., 1994, J. Biol. Chem. 269:28558-28562, which is incorporated herein by reference in its entirety, which may modulate the level of RATH gene expression. Methods for the identification of such intracellular proteins are described, below, in Section 5.4.2.2.

Such intracellular proteins may be involved in the control of immune disorders involving T cell activity, including, but not limited to, TH cell and TH cell subpopulation activation. Such immune disorders include, but are not limited to chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy, and certain neoplasms.

Further, among these compounds are compounds which affect the level of RATH gene expression and/or RATH gene product activity and which can be used in the therapeutic treatment of such immune disorders, as described, below, in Section 5.4.3. Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, and small organic or inorganic molecules.

Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the RATH gene product, and for ameliorating symptoms of immune disorders. Assays for testing the effectiveness of compounds, identified by, for example, techniques such as those described in Section 5.4.2.1-5.4.2.3, are discussed, below, in Section 5.4.2.4. It is to be noted that the compositions of the invention include pharmaceutical compositions comprising one or more of the compounds identified via such methods. Such pharmaceutical compositions can be formulated, for example, as discussed, below, in Section 5.6.

5.4.2.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE RATH GENE PRODUCT

In vitro systems may be designed to identify compounds capable of interacting with, e.g., binding to, the RATH gene products of the invention. Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant RATH gene products, may be useful in elaborating the biological function of the RATH gene product, may be utilized in screens for identifying compounds that disrupt normal RATH gene product interactions, or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that interact with the RATH gene product involves preparing a reaction mixture of the RATH gene product and the test compound under conditions and for a time sufficient to allow the two components to interact with, e.g., bind to, thus forming a complex, which can represent a transient complex, which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring RATH gene product or the test substance onto a solid phase and detecting RATH gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the RATH gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In a particular embodiment of the assays, the RATH gene product utilized comprises the RGS domain contained within the amino acid sequence depicted in FIGS. 1A-1C (SEQ ID NO:2).

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for RATH gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

In a particular embodiment of these assays, the test compound contains at least a portion of a G-α or Gα-I subunit protein such that the assay serves to identify G-α and/or Gα-I subunit amino acid sequences which bind to and form a complex with RATH gene product. The G-α and/or G-α-I subunit sequences tested are preferably derived from G-α and/or G-α-I subunits present in T cells, and are most preferably derived from T cell-specific G-α and/or G-α-I subunits. G-α and/or G-α-I nucleic acid and amino acid sequences are well known to those of skill in the art (see, e.g., Gilman, A., Ann. Rev. Immun., which is incorporated herein by reference in its entirety).

5.4.2.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE RATH GENE PRODUCT

Any method suitable for detecting protein-protein interactions may be employed for identifying RATH protein-intracellular protein interactions.

Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the isolation of intracellular proteins which interact with RATH gene products. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify additional proteins with which it interacts. For example, at least a portion of the amino acid sequence of the intracellular protein which interacts with the RATH gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W. H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening A.; may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result in the simultaneous identification of genes which encode the intracellular protein interacting with the RATH protein. These methods include, for example, probing expression libraries with labeled RATH protein, using RATH protein in a manner similar to the well known technique of antibody probing of λgt11 libraries.

One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the RATH gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, RATH gene products may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait RATH gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait RATH gene sequence, such as the open reading frame of the RATH gene, as depicted in FIGS. 1A-1C can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait RATH gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait RATH gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait RATH gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait RATH gene-interacting protein using techniques routinely practiced in the art.

In a particular embodiment, a two hybrid system can be utilized to identify G-α and/or G-α-I subunit amino acid sequences which bind to RATH gene product. In such an embodiment, RATH "bait" is used in connection with a library encoding G-α and/or G-α-I subunit fragments. G-α and/or G-α-I subunit sequences are preferably derived from G-α and/or G-α-I subunits present in T cells, and are most preferably derived from T cell-specific G-α and/or G-α-I subunits. G-α and/or G-α-I nucleic acid and amino acid sequences are well known to those of skill in the art (see, e.g., Gilman, A., Ann. Rev. Immun., which is incorporated herein by reference in its entirety).

5.4.2.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH RATH GENE PRODUCT/INTRACELLULAR MACROMOLECULE INTERACTION

The RATH gene products of the invention may, in vivo, interact with one or more intracellular macromolecules, such as proteins. Such macromolecules may include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described, above, in Section 5.4.2.2. For purposes of this discussion, such intracellular macromolecules are referred to herein as "interacting partners". Compounds that disrupt RATH interactions in this way may be useful in regulating the activity of the RATH gene product, including mutant RATH gene products. Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in Section 5.4.2.1, above, which would be capable of gaining access to the intracellular RATH gene product.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the RATH gene product and its intracellular interacting partner or partners involves preparing a reaction mixture containing the RATH gene product, and the interacting partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of RATH gene product and its intracellular interacting partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the PATH gene protein and the intracellular interacting partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the RATH gene protein and the interacting partner. Additionally, complex formation within reaction mixtures containing the test compound and normal RATH gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant RATH gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal RATH gene proteins.

The assay for compounds that interfere with the interaction of the RATH gene products and interacting partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the RATH gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the RATH gene products and the interacting partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the RATH gene protein and intracellular interacting partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the RATH gene product or the interacting partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the RATH gene product or interacting partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the interacting components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the RATH gene protein and the interacting partner is prepared in which either the RATH gene product or its interacting partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt RATH gene protein/intracellular interacting partner interaction can be identified.

In a particular embodiment, the RATH gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.1, above. For example, the RATH coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-1, in such a manner that its interacting activity is maintained in the resulting fusion protein. The intracellular interacting partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.2. This antibody can be labeled with the radioactive isotope ¹²⁵ I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-RATH fusion protein can be anchored to glutathione-agarose beads. The intracellular interacting partner can then be added in the presence or absence of the test compound in a manner that allows interaction, e.g., binding, to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the RATH gene protein and the intracellular interacting partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-RATH gene fusion protein and the intracellular interacting partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the RATH gene product/interacting partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In a particular embodiment, one interacting partner is a RATH gene product sequence and the second interacting partner is a G-α-I subunit or the portion of the subunit which binds RATH. Test compounds identified via such an embodiment represent a compound which inhibits RATH/G-α-I subunit complex formation.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the RATH protein and/or the intracellular interacting partner, in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interacting, e.g., binding. Alternatively, one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with, e.g., bind, to its labeled interacting partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the interacting, e.g., binding, domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a RATH gene product can be anchored to a solid material as described, above, in this Section by making a GST-RATH fusion protein and allowing it to bind to glutathione agarose beads. The interactive intracellular binding partner can be labeled with a radioactive isotope, such as ³⁵ S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-RATH fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the intracellular interacting partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.

In another embodiment, a two-hybrid screening assay could be used to identify drugs that block the interaction between RATH and the G-α-1 subunit. This strategy would employ a two-hybrid containing yeast strain whose growth on synthetic complete medium lacking L-histidine is conditional on the physical interaction between RATH and its cognate G-α-1 subunit. In one example of such an embodiment, the strain would be spread in a thin lawn on a plate made of synthetic complete medium lacking L-histidine. Filter disks containing test compounds would be applied to the plates. Most test compounds would not affect the interaction between RATH and its cognate G-α-1 subunit and consequently a confluent lawn of yeast would grow around the disks impregnated with such compounds. Test compounds that inhibit the interaction between RATH and its cognate G-α-1 subunit would block growth of the yeast strain around the filter disks containing them causing zones of growth inhibition. Those compounds could then be tested against wild-type yeast to confirm that they are not simply fungistatic or fungicidal. Such an embodiment can also be performed in liquid culture, utilizing standard well known methods for measuring cell growth in culture.

In a particular embodiment of such methods, one interacting partner is a RATH sequence, while the second interacting partner can be a G-α or G-α-I subunit. In a variation of such an embodiment, the test compound can comprise portions of the G-α or G-α-I subunit such that by identifying inhibitory peptides the method identifies the portion of the subunit which interacts with the RATH gene product.

5.4.2.4. CELL-BASED ASSAYS FOR IDENTIFICATION OF COMPOUNDS WHICH MODULATE RATH ACTIVITY

Cell-based methods are presented herein which identify compounds capable of treating immune disorders by modulating RATH activity. Specifically, such assays identify compounds which affect RATH-dependent G-protein-mediated signal transduction processes. Compounds identified via such methods can, for example, be utilized in methods for treating immune disorders involving T cell, including TH cell and TH cell subpopulation, activity, such as T cell activation.

In one embodiment, the cell-based assays are yeast-based assays comprising yeast cells containing an RATH gene sequence which expresses RATH gene product in the yeast cell. In yeast, RATH, as an RGS family member, modulates signal transduction induced through the phermone-response pathway. In some instances, such modulation represents a "blunting" or desensitization of the signal transduction pathway to external stimulus, e.g., in the case of yeast, a factor mating phermone. Such modulation can easily be scored for in a number of ways (see, e.g., Weiner, J. L. et al., 1993, J. Biol. Chem. 268:8070-8077, which is incorporated herein by reference in its entirety) and can be utilized to identify compounds which modulate RATH activity. That is, such yeast cell-based assays can easily identify test compounds which affect RATH's modulation of signal transduction pathways.

Yeast cells utilized in such assays contain RATH gene sequences, such as those described in Section 5.1 above, capable of being expressed in the yeast cells. The yeast cells can be, for example, mutant yeast cells which are supersensitive to mating phermone, e.g., sst2 loss-of-function mutants such as, for example, sst2 deletion mutants (sst2Δ). Alternatively, wild type yeast cells containing RATH gene sequences capable of being expressed in the yeast cells can be utilized in the assays. Techniques for expressing gene sequences in such yeast cells are well known to those of skill in the art.

Sensitivity of yeast cells to phermone in the presence or absence of test compound can, for example, be tested via the use of growth-arrest (halo) assays. These growth-arrest assays comprise lawns of yeast cells exposed to phermone disks containing increasing levels of yeast phermone. Phermone sensitivity can be assayed by measuring the clear zone which develops around the disks, representing an area of growth inhibition. RATH-containing yeast cells exhibit a decreased sensitivity to phermone and, therefore, the clear zone area around phermone disks should be smaller than that formed by the same type of yeast cells which do not contain RATH.

An assay for identifying compounds which modulate RATH activity can comprise contacting RATH-containing yeast cells as described above with phermone and a test compound for a time sufficient to form a yeast cell lawn, at which time the clear zone area around the phermone source is measured. If the clear zone area differs from the clear zone area surrounding phermone of the same concentration within a lawn produced by the same type of yeast cells in the absence of test compound, then the test compound modulates RATH activity and is a candidate for the treatment of immune disorders.

As a control, a cell of the same yeast type but not containing RATH is exposed to phermone and test compound, in order to identify and discard non RATH-specific modulators.

In instances where the modulatory test compound produces an increase in the clear zone, the test compound inhibits or suppresses RATH activity. In instances where the modulatory test compound produces a decrease in the clear zone, the test compound increases or enhances RATH activity. Depending on the individual immune disorder, either of these two types of modulatory compounds could be utilized as part of a method of treatment.

In an alternative embodiment, yeast cells as described above further containing a phermone-inducible reporter gene can be utilized to identify test compounds which modulate RATH activity. Such phermone-inducible reporter genes are well known to those of skill in the art and can include, for example, a calorimetric reporter sequence e.g., lacZ, whose expression is phermone-inducible (see, e.g., Druey, K. M. et al., 1996, Nature 379:742-746).

In such an assay, the yeast cells are contacted with test compound and phermone. Reporter gene expression in the presence of phermone is measured in the presence and absence of test compound. If reporter gene expression in the presence of test compound differs, the test compound modulates RATH activity and is a candidate for the treatment of immune disorders.

As a control, a cell of the same yeast type but not containing RATH is exposed to phermone and test compound, in order to identify and discard non RATH-specific modulators.

In instances where the modulatory test compound produces an increase in reporter gene expression, the test compound inhibits or suppresses RATH activity. In instances where the modulatory test compound produces a decrease in reporter gene expression, the test compound increases or enhances RATH activity. Depending on the individual immune disorder, either of these two types of modulatory compounds could be utilized as part of a method of treatment.

In an alternate embodiment, expression cloning of mammalian G-protein coupled receptor pathway inhibitors may be carried out in yeast cells containing a RATH gene sequence which expresses RATH gene product in the yeast cell. Binding of mating phermone such as alpha-factor to its receptor arrests cell growth. A MATa yeast strain that expresses the alpha factor receptor can be transformed with a library of murine or human cDNAs cloned in a yeast expression vector. Yeast transformants are then plated on a growth medium that selects for transformants and contains a concentration of mating phermone known to inhibit the growth of wild-type yeast cells. Transformants that are able to grow are isolated, and the library plasmid that they contain, is isolated. In order to eliminate false positives, isolated plasmids are reintroduced into the original MATa strain and the above screen is repeated. Those yeast cells that are able to grow contain a plasmid able to confer resistance to mating phermone on the host strain. cDNAs isolated in this fashion are sequenced and biologically characterized.

5.5. METHODS FOR THE AMELIORATION OF T CELL DISORDER SYMPTOMS

Described below are methods and compositions for treating immune disorders and immune disorder symptoms. Such immune disorders can include, but are not limited to, chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.

Such methods comprise methods which modulate RATH gene and gene product activity. In certain instances the treatment will require an increase, upregulation or activation of RATH activity, while in other instances, the treatment will require a decrease, downregulation or suppression of RATH activity. "Increase" and "decrease" refer to the differential level of RATH activity relative to RATH activity in the cell type of interest in the absence of modulatory treatment. Methods for the decrease of RATH activity are discussed in Section 5.5.1, below. Methods for the increase of RATH activity are discussed in Section 5.5.2, below. Methods which can either increase or decrease RATH activity depending on the particular manner in which the method is practiced are discussed in Section 5.5.3, below.

5.5.1 METHODS FOR DECREASING RATH ACTIVITY

As discussed, above, successful treatment of certain immune disorders can be brought about by techniques which serve to decrease RATH activity. Activity can be decreased by, for example, directly decreasing RATH gene product activity and/or by decreasing the level of RATH gene expression.

For example, compounds such as those identified through assays described, above, in Section 5.4, above, which decrease RATH activity can be used in accordance with the invention to ameliorate symptoms associated with such immune disorders. As discussed in Section 5.4, above, such molecules can include, but are not limited to peptides, including soluble peptides, and small organic or inorganic molecules, and can be referred to as RATH antagonists. Techniques for the determination of effective doses and administration of such compounds are described, below, in Section 5.6.

Further, antisense and ribozyme molecules which inhibit RATH gene expression can also be used in accordance with the invention to reduce the level of RATH gene expression, thus effectively reducing the level of RATH gene product present, thereby decreasing the level of RATH activity. Still further, triple helix molecules can be utilized in reducing the level of RATH gene activity. Such molecules can be designed to reduce or inhibit either wild type, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to RATH gene mRNA. The antisense oligonucleotides will bind to the complementary RATH gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary to either the 5'- or 3'- non-translated, non-coding regions of the RATH gene, as shown, for example, in FIGS. 1A-1C, could be used in an antisense approach to inhibit translation of endogenous RATH gene mRNA.

Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of target or pathway gene mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

The antisense molecules should be delivered to cells which express the RATH gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous RATH gene transcripts and thereby prevent translation of the RATH gene mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (For a review see, for example Rossi, J., 1994, Current Biology 4:469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety. As such, within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.

Ribozyme molecules designed to catalytically cleave RATH gene mRNA transcripts can also be used to prevent translation of RATH gene mRNA and expression of target or pathway gene. (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy RATH gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the RATH gene mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in an RATH gene.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the RATH gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous RATH gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

In instances wherein the antisense, ribozyme, and/or triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique can also efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility can arise wherein the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, therefore, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, in instances whereby the target gene encodes an extracellular protein, it can be preferable to coadminister normal target gene protein in order to maintain the requisite level of target gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Endogenous RATH gene expression can also be reduced by inactivating or "knocking out" the target and/or pathway gene or its promoter using targeted homologous recombination. (E.g., see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell 5:313-321; each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional RATH gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous RATH gene (either the coding regions or regulatory regions of the RATH gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express RATH gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the RATH gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive RATH gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). Such techniques can also be utilized to generate immune disorder animal models. It should be noted that this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, e.g., herpes virus vectors.

Alternatively, endogenous RATH gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the RATH gene (i.e., the RATH gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the RATH gene in target cells in the body. (See generally, Helene, C. 1991, Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y. Accad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

5.5.2. POSITIVE MODULATORY TECHNIQUES

As discussed above, successful treatment of certain immune disorders can be brought about by techniques which serve to increase the level of RATH activity. Activity can be increased by, for example, directly increasing RATH gene product activity and/or by increasing the level of RATH gene expression.

For example, compounds such as those identified through assays described, above, in Section 5.4, which increase RATH activity can be used in accordance with the invention to treat immune disorders and immune disorder symptoms. As discussed in Section 5.4, above, such molecules can include, but are not limited to peptides, including soluble peptides, and small organic or inorganic molecules, and can be referred to as RATH antagonists.

For example, a compound can, at a level sufficient to treat immune disorder and symptoms, be administered to a patient exhibiting such symptoms. Any of the techniques discussed, below, in Section 5.6, can be utilized for such administration. One of skill in the art will readily know how to determine the concentration of effective, non-toxic doses of the compound, utilizing techniques such as those described, below, in Section 5.6.1.

In instances wherein the compound to be administered is a peptide compound, DNA sequences encoding the peptide compound can be directly administered to a patient exhibiting immune disorder symptoms, at a concentration sufficient to produce a level of peptide compound sufficient to ameliorate symptoms of an immune disorder, for example, multiple sclerosis. Any of the techniques discussed, below, in Section 5.6, which achieve intracellular administration of compounds, such as, for example, liposome administration, can be utilized for the administration of such DNA molecules. The DNA molecules can be produced, for example, by well known recombinant techniques.

In the case of peptides compounds which act extracellularly, the DNA molecules encoding such peptides can be taken up and expressed by any cell type, so long as a sufficient circulating concentration of peptide results for the elicitation of a reduction in the immune disorder symptoms. In the case of compounds which act intracellularly, the DNA molecules encoding such peptides must be taken up and expressed by the T cell population of interest at a sufficient level to bring about the reduction of immune disorders.

Any technique which serves to selectively administer DNA molecules to the T cell population of interest is, therefore, preferred, for the DNA molecules encoding intracellularly acting peptides, via, for example a delivery complex.

Such a delivery complex can comprise an appropriate nucleic acid molecule and a targeting means. Such targeting means can comprise, for example, sterols lipids, viruses or target cell specific binding agents. Viral vectors can include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. In the case of asthma, for example, techniques for the selective administration of the molecules to T cell subpopulations residing within lung tissue are preferred.

Further, in instances wherein the TH cell subpopulation-related disorder involves an aberrant RATH gene, patients can be treated by gene replacement therapy. One or more copies of a normal RATH gene or a portion of the gene that directs the production of a normal RATH gene protein with RATH gene function, can be inserted into cells, via, for example a delivery complex.

Such a delivery complex can comprise an appropriate nucleic acid molecule and a targeting means. Such targeting means can comprise, for example, sterols lipids, viruses or target cell specific binding agents. Viral vectors can include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. For example, such RATH gene sequences can encode peptide sequences which comprise the RATH RGS domain contained within the amino acid sequence depicted in FIGS. 1A-1C (SEQ ID NO:2). Such gene replacement techniques can be accomplished either in vivo or in vitro. As the RATH gene encodes a gene product which acts intracellularly, the gene must be expressed with the T cell population of interest. Techniques which select for expression within the cell type of interest are, therefore, preferred. In vivo, such techniques can, for example, include appropriate local administration of RATH gene sequences.

Additional methods which may be utilized to increase the overall level of RATH activity include the introduction of appropriate RATH gene-expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of the immune disorder of interest. Such cells may be either recombinant or non-recombinant. Among the cells which can be administered to increase the overall level of RATH gene expression in a patient are normal cells, which express the RATH gene. The cells can be administered at the anatomical site of expression, or as part of a tissue graft located at a different site in the body. Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, et al., U.S. Pat. No. 5,399,349; Mulligan & Wilson, U.S. Pat. No. 5,460,959.

In vitro, RATH gene sequences can be introduced into autologous cells. These cells expressing the RATH gene sequence of interest can then be reintroduced, preferably by intravenous administration, into the patient until the disorder is treated and symptoms of the disorder are ameliorated.

5.5.3 ADDITIONAL MODULATORY TECHNIQUES

Described herein are modulatory techniques which, depending on the specific application for which they are utilized, can yield either increase or decrease RATH activity levels leading to the amelioration of immune disorders such as those described above.

Antibodies exhibiting modulatory capability can be utilized to ameliorate immune disorders. Depending on the specific antibody, the modulatory effect can be increase or decrease RATH activity. Such antibodies can be generated using standard techniques described in Section 5.3, above, against full length wild type or mutant RATH proteins, or against peptides corresponding to portions of the proteins. The antibodies include but are not limited to polyclonal, monoclonal, FAb fragments, single chain antibodies, chimeric antibodies, and the like.

Because RATH is an intracellular protein, it is preferred internalizing antibodies be used. However, lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region which binds to the RATH gene product epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment which binds to the RATH protein's binding domain is preferred. For example, peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the RATH protein can be used. Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g., see Creighton, 1983, supra; and Sambrook et al., 1989, above). Alternatively, single chain antibodies, such as neutralizing antibodies, which bind to intracellular epitopes can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Marasco, W. et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

5.6. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The compounds and nucleic acid sequences described herein can be administered to a patient at therapeutically effective doses to treat or ameliorate immune disorders, e.g., TH cell-related disorders. A therapeutically effective dose refers to that amount of a compound or TH cell population sufficient to result in amelioration of the immune disorder symptoms, or alternatively, to that amount of a nucleic acid sequence sufficient to express a concentration of RATH gene product which results in the amelioration of the immune disorder symptoms.

5.6.1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

5.6.2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts and solvents can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration (i.e., intravenous or intramuscular) by injection, via, for example, bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. It is preferred that the TH cell subpopulation cells be introduced into patients via intravenous administration.

The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

6. EXAMPLE IDENTIFICATION OF A NOVEL GENE FAMILY (RATH) INVOLVED IN T CELL ACTIVITY

Described herein is the identification and characterization of a novel gene family referred to as the RATH gene family. Members of the RATH gene family contain RGS ("regulator of G-protein signalling") domains, exhibit a T cell, TH cell or TH cell subpopulation specific or enhanced expression pattern, and can be involved in modulation of T cell activity, such as T cell activation. The identification, characterization of an exemplary RATH gene family member, termed RATH1.1, is described below.

6.1. PARADIGMS FOR THE IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED GENES IN TH CELL SUBPOPULATIONS

The differential display technique is a procedure, utilizing the well-known polymerase chain reaction (PCR; the experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), which allows for the identification of sequences derived from genes which are differentially expressed. As described below in Section 6.2, such a technique was utilized for the identification of gene sequences which are involved in immune disorder states, e.g., TH cell subpopulation-related disorder states, and/or which are involved in differentiation, maintenance and/or effector function of TH cell subpopulations.

Briefly, TH cell subpopulations activated with appropriate antigens after which the expression patterns of, e.g. TH 1 vs. TH2 and/or activated vs. non-activated cells were analyzed. As described in detail in Section 6.2, activated and non-activated TH1 and TH2 cell lines were analyzed according to such a method.

After stimulation, RNA is isolated and reverse-transcribed into single-stranded cDNA, utilizing standard techniques which are well known to those of skill in the art. Next, pairs of PCR primers, as described below, which allow for the quantitative amplification of clones representing a reproducible subset of the RNA transcripts present within any given cell. Utilizing different pairs of primers allows each of the primed mRNA transcripts present in a cell to be amplified. Among such amplified transcripts can be identified those which have been produced from differentially expressed genes.

The pattern of clones resulting from the reverse transcription and amplification of the mRNA of two different cell types is displayed and compared via sequencing gel electrophoresis. Differentially expressed genes are indicated by differences in the two banding patterns. Once identified the band can be purified and used as a probe to screen a library for full length cDNA clones. Such a technique was successfully utilized, as described below, to identify the RATH 1.1 gene.

6.2 ISOLATION OF AN INDUCIBLE TH1 SPECIFIC GENE SEQUENCE

The section below describes the identification of a novel gene sequence differentially expressed in a TH1-specific manner.

6.2.1 STIMULATION, CELL CULTURE AND PREPARATION OF TH1 AND TH2 RNA

To identify gene sequences differentially expressed in TH cell subpopulations, the differential display technique, described above, in Section 6.1, was utilized. TH1 and TH2 clonal cell lines were stimulated, and RNA from the cell lines was isolated. Two TH1 (D1.1 and Dorris) and two TH2 (Dax and CDC25) cell lines were utilized.

For normal culture, T-helper cells were stimulated every 10-14 days with freshly isolated MHC-matched antigen presenting cells, in combination with specific antigen (D1.1: Rabbit Gamma Globulin, Sigma; Dorris: HEL lysozyme, Sigma; Dax: Hel lysozyme, Sigma; CDC25: Rabbit Gamma Globulin, Sigma). Further, cultures were fed every 2-3 days with fresh IL-2 (Boehringer Mannheim). Cells were cultured according to standard protocols (Coligan, et al., eds., 1995, Current Protocols in Immunology, John Wiley & Sons, Inc.).

Prior to stimulation, cells were starved of both antigen (at least 10 days) and IL-2 (2-3 days), and viable cells were purified with Lymphocyte Separation Medium (Organon Teknika). Cells were separated into two groups. One group was left unstimulated. One group was stimulated for 6 hours by plating on plastic culture dishes which had been coated with anti-mouse CD3 epsilon monoclonal antibody (hybridoma supernatent from the 145- 2C11 hybridoma; Parmingen, Inc., San Diego Calif.) at 1.5 μg/ml in PBS for one hour at 37° C.

Total cellular RNA was isolated from the cells of each group (unstimulated or anti-CD3 stimulated) as follows. Specifically, cells were quick frozen on dry ice, homogenized together with a mortar and pestle under liquid nitrogen and total cellular RNA was extracted from cells with RNAzol™ (TEL-TEST, Inc., Friendswood Tex.) according to the manufacturer's instructions. Briefly, the cells were solubilized in an appropriate amount of RNAzol™ and RNA was extracted by the addition of 1/10 v/v chloroform to the solubilized sample followed by vigorous shaking for approximately 15 seconds. The mixture was then centrifuged for 15 minutes at 12,000 g and the aqueous phase was removed to a fresh tube. RNA was precipitated with isopropanol. The resultant RNA pellet was dissolved in water and re-extracted with an equal volume of chloroform to remove any remaining phenol. The extracted volume was precipitated with 2 volumes of ethanol in the presence of 150 mM sodium acetate. The precipitated RNA was dissolved in water and the concentration determined spectroscopically (A₂₆₀).

Residual DNA was removed by digesting for 30 min at 37° C. with RNAse free DNAse I (Boehringer Mannheim). After extraction with phenol/chloroform and ethanol precipitation, the RNA was dissolved in DEPC (diethyl pyrocarbonate)-treated water.

RNA populations from the two different stimulated and unstimulated TH1 and TH2 cell lines were compared using the Delta RNA Fingerprinting Kit (Clontech #PT1173-1), according to the manufacturer's protocol.

6.2.2 IDENTIFICATION OF A TH1 SPECIFIC GENE SEQUENCE

The RNA isolated for TH1 and TH2 cells, as described, above, in Section 6.2.1. , was quantitatively amplified and the cells' gene expression patterns were analyzed as described herein. The analysis yielded an approximately 220 bp differentially expressed cDNA gene sequence which, interestingly, was expressed exclusively in the two TH1 clones examined, but was absent in non-stimulated TH1 clones and in all the TH2 clones examined.

Specifically, cDNA synthesis was performed using DNA-free RNA (above) from 2 independent TH1 clones and 2 independent TH2 clones. For each sample, cDNA synthesis was performed using 2 μg of total RNA and 1 μl of oligo dT primer (1 μM), as described in the manufacturer's protocol. cDNA samples were stored at -20° C., and used for PCR amplification using various combinations of the following 25 bp 5' (P primers) and 30 bp 3' (T primers):

P: 5'-ATTAACCCTCACTAAATGCTGGGTG-3' (SEQ ID NO:3) (forward primer) and

T: 5'-CATTATGCTGAGTGATATCTTTTTTTTTAA-3' (SEQ ID NO:4) (reverse primer).

PCR amplification was performed according to the provided protocol, using the CLONTECH Advantage cDNA PCR Core Kit (K1905-1). Three low stringency PCR cycles (annealing temp. 40° C.) were followed by 22-25 high stringency PCR cycles (annealing temp. 60° C.), and PCR products were separated by denaturing PAGE using 5% polyacrylamide/8M urea gels, and PCR products were visualized by autoradiography. Differentially expressed bands were excised from dried gels and re-amplified using the original primers. Re-amplified products were cloned into pCR 2.1, using the TA Cloning Kit (Invitrogen), and individual isolates were sequenced.

6.3 CLONING AND CHARACTERIZATION OF RATH1.1, A NOVEL TH1-SPECIFIC GENE

The subsection below describes the isolation and characterization of a gene, termed the RATH1.1 gene. The RATH1.1 gene was obtained by screening of a TH1-specific cDNA library with the 220 bp cDNA fragment described above, in Section 6.2. Sequence analysis reveals that RATH1.1 is a novel gene encoding a product which contains an RGS domain. Expression analysis confirms that RATH1.1 represents a TH1-specific gene, and can be involved in T cell activity. RATH1.1 appears to represent a member of a novel gene family, termed the RATH gene family, whose members contain RGS domains, are expressed in a T cell-restricted manner and can be involved in T cell activity such as, for example, T cell activation.

5 6.3.1 PREPARATION AND SCREENING OF TH-1 SPECIFIC LIBRARY

A TH1-specific cDNA library was screened in an attempt to obtain the full-length sequence corresponding to the 220 bp sequence identified in Section 6.2, above. A TH1-specific cDNA library was generated using polyA+ RNA isolated from the TH1 cell line Dorris (anti-CD3 stimulated), using the Fast Track 2.0 Kit (Invitrogen). cDNA was generated using oligo dT primers and cloned into lambda ZIPLOX phage arms (GIBCO BRL 15394-018), using the Sal I and Not I cloning sites.

The TH-1 specific phage library was screened using a labeled probe generated from the re-amplified 220 bp fragment obtained by the differential display method described above in Section 6.2. Briefly, plaques were screened with the probe overnight at 65° C. in Church's buffer (7% SDS, 250 mM NaHPO₄, 3 μM EDTA, 1% BSA). The next day, filters were washed in 2×SSC/1% SDS for 30 minutes at 50° C. Positive plaques were rescreened under the same conditions.

The largest positive clone obtained was 2278 bp, and contained one complete open reading frame, which codes for a 201 amino acid having a predicted molecular weight of 22.6 kD. The gene/protein is now referred to as RATH1.1. The RATH1.1 nucleotide and amino acid sequences are depicted in FIGS. 1A-1C.

Comparison of the RATH1.1 sequence to known genes (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410 indicates that RATH1.1 is a novel member of the RGS (regulator of G-protein signalling) family, a gene family thought to encode products involved in G protein-mediated signal transduction (see, e.g., Druey, K. M. et al., 1996, Nature 379:742-746). For example, RATH1.1 exhibits approximately 50% identity and 70% similarity to the RGS4 gene product. The RGS domain is located from approximately amino acid residue 60 to amino acid residue 179 of the R11 amino acid sequence shown in FIGS. 1A-1C.

6.3.1. EXPRESSION OF RATH GENES

Northern blot analysis was carried out to determine the size of the RATH1.1 transcript and the tissue distribution of RATH1.1 expression. As described below, the expression analysis confirmed the TH1-specific RATH1.1 gene expression pattern and, further, revealed that the gene produces a transcript of approximately 3 kb.

For northern analysis of murine tissues, total cellular RNA from various murine tissues (spleen, thymus, lymph node, muscle, heart, liver, kidney, brain, uterus and testes) was isolated using the RNA purification techniques as described above in Section 6.2. Cell lines tested included the following: WEHI-3B myelomonocyte, Pu5-1.8 myelomonocyte, P388D1 monocyte-macrophage, IC-21 macrophage, AKR.G2 thyoma, BaF3 pro B-cell, EL-4 lymphoma, NFS-1.0 C-1 B-cell lymphoma, SOT embryonic fibroblast, EOMA endothelial and BMS-12 bone marrow.

Stimulated and unstimulated TH1 clones D1.1, Dorris and AE7, and TH2 clones D10.G4, DAX and CDC25 were also tested. The TH-1 and TH-2 clones were cultured and stimulated as described above in Section 6.2.1.

RNA samples were electrophoresed in a denaturing agarose gel containing 1-1.5% agarose (SeaKem™ LE, FMC BioProducts, Rockland, Me.) containing 3% formaldehyde. For northern analysis of hematopoietic cell lines, a total of approximately 20 μg of total RNA was loaded per lane, for all other cell lines approximately 15 μg of total RNA was loaded per lane. Samples containing the indicated amounts of total RNA were mixed with denaturing loading solution (72% deionized formamide and bromophenol blue) and heated to 70° C. for 5 minutes. Samples were placed on ice and immediately loaded onto gels. Gels were run in 1× MOPS buffer (100 mM MOPS, 25 mM sodium acetate, 5 mM EDTA). After electrophoresis, the gels were stained with ethidium bromide and visualized with ultraviolet light.

After completion of electrophoresis, gels were soaked in 50 mM sodium hydroxide with gentle agitation for approximately 30 minutes to lightly cleave RNA. Gels were rinsed twice in water and then neutralized by soaking in 0.1M Tris-HCl (pH 7.5) for approximately 30 minutes. Gels were briefly equilibrated with 20×SSC (3M sodium chloride, 0.3M sodium citrate) and then transferred to nylon membranes such as Hybond™, -N, (Amersham, Inc., Arlington Heights, Ill.) or Zeta-Probe (Bio-Rad, Inc., Hercules, Calif.) overnight in 20×SSC. Membranes containing transferred RNA were baked at 80° C. for 2 hours to immobilize the RNA.

The 220 bp RATH1.1 probe was prepared as follows. Briefly, 50 ng of purified DNA fragment was used to generate the probe. The fragment was added to a 20 μl random hexanucleotide labeling reaction (Boehringer Mannhein, Inc., Indianapolis, Ind.) containing random hexamers and a mix of the nucleotides dCTP, dGTP, and dTTP (at a final concentration of 25 μM each). The reaction mix was heat-denatured at 100° C. for 10 minutes and then chilled on ice. 5 μl of α-³² P-ATP (50 μCi; Amersham, Inc., Arlington Heights, Ill.) and Klenow DNA polymerase (2 units; Boehringer Mannheim, Inc., Indianapolis, Ind.) were added. Reactions were incubated at 37° for 30 minutes. Following incubation, 30 μl water was added to the labeling reaction and unincorporated nucleotides were removed by passing the reactions through a BioSpin-6™ chromatography column (Bio-Rad, Inc., Hercules, Calif.). Specific incorporation was determined using a scintillation counter. 1-5×10⁶ cpm were used per ml hybridization mixture.

Nylon membranes containing immobilized RNA were prehybridized according to manufacturer's instructions. Radiolabelled probes were heat denatured at 70° C. in 50% deionized formamide for minutes and then added to the hybridization mixture (containing 50% formamide, 10% dextran sulfate, 0.1% SDS, 100 μg/ml sheared salmon sperm DNA, 5×SSC, 5× Denhardt's solution, 30mM Tris-HCl (pH 8.5), 50 mM NaPO₄ (pH 5). Hybridizations were carried out at 42° C. overnight. Nylon membranes were then bathed for 2 minutes in a wash solution of 0.2×SSC and 0.1% SDS at room temperature to remove most of the remaining hybridization solution. The membranes were then bathed twice in fresh 42° C. preheated wash solution for 20 minutes. Filters were covered in plastic wrap and exposed to autoradiographic film to visualize results.

Northern Blot analyses verify that the RATH1.1 gene is highly upregulated in stimulated TH1 cells. First, northern blot analysis of 3 individual TH1 clones and 3 individual TH2 clones revealed an approximately 3 kb RNA which is highly up-regulated in stimulated TH1 clones, but is absent from all other samples tested.

Consistent with the cell clone data, RATH1.1 message appears to be absent from all of the murine tissues analyzed (i.e., spleen, thymus, lymph node, muscle, heart, liver, kidney, brain, uterus and testes), although the RATH1.1 probe clearly hybridized with itself. Third, northern blot analysis was also performed with a panel of murine hematopoietic cell lines. RNA was obtained from various cell lines which had either been left unstimulated or had been stimulated with appropriate antigen (i.e., either PMA or LPS, as described, above, in Section 1). While RATH1.1 RNA was detected in several of the cell lines tested (Pu51.8, STO, EOMA and BMS12), the expression level was significantly lower than was seen in anti-CD3 stimulated TH1 cells (i.e., only a faint signal was detectable after a 3 day exposure).

6.4. CHROMOSOMAL MAPPING OF THE RATH 1.1 GENE

To determine the chromosomal map position of the RATH1.1 gene the technique of Single Stranded Conformational Polymorphism (SSCP) gel electrophoresis was used. More specifically, the genetic segregation of the Mus spretus allele of RATH1.1 was followed in 188 backcross progeny of cross (C57B1/6J×Mus spretus) F₁ females×C57B1/6J males.

PCR primers were designed from the coding sequence of RATH1.1 cDNA. The following RATH1.1 derived PCR primers were used for amplification of mouse genomic DNA:

5'-AGGCAAGGGGCAGGAAATGAAG-3' (SEQ ID NO:5) (forward primer)

5'-ACTAAATGCTGGGTGGTTGG-3' (SEQ ID NO:6) (reverse primers).

The PCR reaction mixture contained 6μl template DNA (10 ng/μl), 1.4μl 10× Perkin Elmer (Norwalk, Conn.) PCR buffer, 1.12 μl dNTPs (2.5 mM), 1.05 μl Forward primer (6 μM), 1.05 μl Reverse primer (6 μM), 0.38 μl H₂ O and 3 μl Amplitaq Hotstart™ polymerase (Perkin Elmer; 0.5U/μl).

The amplification profile was as follows: 94° C., 2 minutes, at which point the Amplitaq™ was added; then 30 cycles of 94° C., 40 seconds; 55°, 50 seconds; and 72° C., 30 seconds.

Samples were run on both (a) nondenaturing 8% acrylamide gels run at 45 W, room temperature, for 3 hours and (b) nondenaturing 10% acrylamide SSCP (single stranded conformational polymorphism) gels run at 20 W, 4° C., for 2.5 hours. Both types of gels were stained with SYBR Green I and scanned on an MD fluorimager, and gave interpretable results. The primers amplified a 159 bp fragment from C57B1/6J Mus spretus genomic DNA, consistent with the base pair length between the two primers in the RATH1.1 cDNA.

The segregation pattern of the Mus spretus allele was compared to the segregation pattern of 226 other genetic loci that have been mapped in this backcross panel. By minimizing the number of multiple crossovers between RATH1.1 and other markers it was determined that RATH1.1 maps to murine chromosome 1, 9.8 cM distal of D1MIT12 and 29.2 cM proximal of D1MIT17 (two MIT microsatellite markers; Research Genetics).

6.5 RATH1.1 MODULATES G-PROTEIN MEDIATED SIGNAL TRANSDUCTION

Mutations in the sst2 gene, which encodes an RGS-containing gene product, cause increased sensitivity and defective desensitization to signal transduction induced by alpha factor mating pheromone. The results described below demonstrate complementation of sst2 mutations in yeast by the RATH1.1 protein. Thus, these results prove that RATH1.1 acts to modulate G protein-mediated signal transduction. Functional complementation of an sst2 mutation by RATH1.1 provides a convenient yeast-based bioassay for RATH1.1 function. Such a bioassay can, for example, be used to identify molecules that are agonists or antagonists of RATH1.1.

Plasmids containing the RATH1.1 coding sequence were constructed as follows. The RATH1.1 coding sequence was cloned into yeast expression vectors pYADE4 and pYPGE2. pYADE4 contains the ADH2 promoter and pYPGE2 contains the PGK promoter. On media where glucose is the sole carbon source, such as the media used in this experiment, the PGK promoter is reported to be 17 times stronger than the ADH2 promoter (Brunelli and Pall, 1993, Yeast, 9:1299-1308).

Oligonucleotides D1 (5'-AGC GGA TCC AAA AAA ATG ACC ATG ATT ACG CCA AGO TCT AAT AC-3') (SEQ ID NO:7) and D2 (5'-AGC AAT CGA TGG AGG CTC AAG TGT GTG AAG GCT CAG-3') (SEQ ID NO:8) were used to amplify the entire predicted 606 base pair coding sequence of RATH1.1 predicted to encode a 201 (plus stop codon) amino acid protein. Restriction endonuclease sites BamHI and ClaI were engineered into the oligonucleotides to allow the cloning of the PCR fragment encoding the RATH1.1 protein into yeast expression vectors pYADE4 and pYPGE2 (Brunelli and Pall, 1993, Yeast, 9:1299-1308). In addition, the sequence AAAAAA was introduced immediately upstream of the initiator ATG codon to promote efficient translation in yeast (Hinnebusch and Liebman, 1991, The Molecular Biology and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Cold Spring Harbor Laboratory Press, 627-735). The PCR product coding RATH1.1 was doubly digested with BamHI and ClaI and ligated after agarose gel purification into pYADE4 and pYPGE2 doubly digested with BamHI and ClaI. Ligation reactions were transformed into the DH5α E. coli stain (Gibco BRL catalog). One E. coli clone containing the RATH1.1 coding sequence in pYADE4 was isolated and named pMB135 and one E. coli clone containing the RATH1.1 coding sequence in pYPGE2 was isolated and named pMB136.

Standard yeast media including synthetic complete medium lacking L-tryptophan were prepared and yeast genetic manipulation were performed as described (Sherman, 1991, Meth. Enzymol., 194:3-21). Yeast transformations were performed using standard protocols (Gietz et al., 1992, Nucleic Acids Res., 20:1425. Ito et al, 1983, J. Bacteriol., 153:163-168).

Isogenic SST2⁺ (MF42) and sst2⁻ (MF41) strains were transformed with pYADE4, pYPGE2, pMB135, and pMB136. Transformants were selected on synthetic complete medium lacking L-tryptophan. Transformants were picked from each transformation and were used for further experimentation. The transformants were designated as follows:

MF41 transformed with pYADE4 (MF167); MF41 transformed with pYPGE2 (MF168); MF41 transformed with pMB135 (MF169); MF41 transformed with pMB136 (MF170); MF42 transformed with pYADE4 (MF171); MF42 transformed with YPGE2 (MF172); MF42 transformed with pMB135 (MF173); and MF42 transformed with pMB136 (MF174).

The MF41 and MF42 transformants were subjected to desensitization assays. The assays were performed as follows. MF41 and MF42 transformants were grown to saturation overnight in liquid synthetic complete medium lacking L-tryptophan. Each culture was diluted 1:40 into 2 mls of liquid synthetic complete medium lacking L-tryptophan which was added to 2 mls of 1% (wt/vol) agar dissolved in water and warmed to 50° c. The resulting cell suspension was plated on a synthetic complete medium lacking L-tryptophan plate and allowed to solidify. Sterile BBL blank paper 1/4 inch disks (BBL Microbiology Systems) were spotted with 10, 1, 0.1, or 0.01 mg of synthetic alpha factor (Sigma) and were arrayed on top of the solidified agar medium. The diameter of the halos of growth arrested cells surrounding the filter disks containing alpha factor were photographed and measured after 24 hours of growth.

The results obtained from the desensitization assays are tabulated in Table 1. 10 μg of alpha factor causes the SST2+ strain transformed with either pYADE4 or pYPGE2 (MY171 and MY172) to have a ≈1.8 cm zone of growth inhibition. 10 μg of alpha factor causes the SST2+ strain expressing RATH1.1 under the control of the ADH2 promoter (MY173) to have a 1.6 cm zone of growth inhibition and the SST2+ strain expressing RATH1.1 under the control of the strong PGK promoter (MY174) to have a no zone of growth inhibition. RATH1.1 expressed from the strong PGK promoter dramatically negatively regulates the pheromone response pathway and in the assay used in this study completely prevents the signal created by 10 μg of alpha factor from being transduced. Because RATH1.1 is an RGS protein and RGS proteins are thought to function as GAPs (GTPase Activating proteins), it is likely the inhibition of pheromone signal transduction caused by high level RATH1.1 expression in yeast is due to RATH1.1 acting as a GAP on the GPA1 protein, the G-protein alpha subunit that is coupled to the alpha factor receptor in yeast.

10 μg of alpha factor causes the sst2- strain transformed with either pYADE4 or pYPGE2 (MY167 and MY168) to have a ≈3.7 cm zone of growth inhibition. 10 μg of alpha factor causes the sst2- strain expressing RATH1.1 under the control of the ADH2 promoter (MY169) to have a 1.8 cm zone of growth inhibition which is similar to the ≈1.8 cm zone of growth inhibition that 10 μg of alpha factor elicits in a SST2+ strain (MY171 and MY172). Consequently RATH1.1 under the control of the ADH2 promoter functionally complements the sst2- mutation. Such complementation of an sst2- mutation by RATH1.1 is strong evidence that RATH1.1 has GAP activity towards GPA1 and more generally towards G-protein alpha subunits.

10 μg of alpha factor causes the sst2- strain expressing RATH1.1 under the control of the strong PGK promoter (MY170) to have no zone of growth inhibition. The experimental results indicate that in the absence of the SST2 protein, the RATH1.1 protein can completely block the transduction of the signal produced by 10 μg of alpha factor and supports the idea that RATH1.1 acts as a GAP on GPA1. This experimental results also indicate that the alpha factor resistance phenotype conferred by PATH1.1 expression plasmids is a function of the level of RATH1.1 expression, i.e., the stronger PGK promoter causes a stronger phenotype than the weaker ADH2 promoter.

                  TABLE 1                                                          ______________________________________                                                   Amount of alpha factor added to disk                                 Strain Name 10 μg                                                                              1 μg    1 μg                                                                             .01 μg                                   ______________________________________                                         MY167       3.7    2.7        1.7  1.0                                         MY168       3.8    2.7        1.7  1.0                                         MY169       1.8    0.8        None None                                        MY170       None   None       None None                                        MY171       1.8    0.9        None None                                        MY172       1.9    0.8        None None                                        MY173       1.6    None       None None                                        MY174       None   None       None None                                        ______________________________________                                          Diameters of growth inhibition zones in cm caused by filter disks              impregnated with different quantities of alpha factor are shown, in            centimeters, below each alpha factor concentration.                      

7. DEPOSIT OF MICROORGANISMS

The following microorganism was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on Jul. 30, 1996 and assigned the indicated accession number:

    ______________________________________                                         Microorganism  ATCC Accession No.                                              ______________________________________                                         E. coli, femtR11A                                                                             98116                                                           ______________________________________                                    

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 8                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2272 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 41..646                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CCACGCGTCCGCCACGACGTGCTGTCCTGCGTCCGCAACCATGTGCCGCACCCTA55                      MetCysArgThrLeu                                                                15                                                                             GCCACCTTCCCCAACACCTGCCTGGAGAGAGCCAAAGAGTTCAAGACG103                            AlaThrPheProAsnThrCysLeuGluArgAlaLysGluPheLysThr                               101520                                                                         CGGCTGGGAATCTTTCTTCATAAATCAGAGCTGAGCTCCGATACTGGG151                            ArgLeuGlyIlePheLeuHisLysSerGluLeuSerSerAspThrGly                               253035                                                                         GGTATTAGCAAATTCGAGTGGGCCAGTAAGCATAACAAAGAGAGAAGC199                            GlyIleSerLysPheGluTrpAlaSerLysHisAsnLysGluArgSer                               404550                                                                         TTCTCAGAAGATGTACTGGGATGGAGAGAGTCTTTCGATTTGCTGCTG247                            PheSerGluAspValLeuGlyTrpArgGluSerPheAspLeuLeuLeu                               556065                                                                         AACAGTAAAAATGGGGTGGCTGCCTTCCATGCCTTCCTAAAGACGGAA295                            AsnSerLysAsnGlyValAlaAlaPheHisAlaPheLeuLysThrGlu                               70758085                                                                       TTCAGTGAGGAGGCGCTGGAGTTCTGGTTGGCCTGCGAGGAGTTCAAG343                            PheSerGluGluAlaLeuGluPheTrpLeuAlaCysGluGluPheLys                               9095100                                                                        AAGATCCGATCAGCCACCAAACTGGCGTCCAGGGCTCACCACATCTTT391                            LysIleArgSerAlaThrLysLeuAlaSerArgAlaHisHisIlePhe                               105110115                                                                      GACGAGTACATCCGCAGCGAAGCCCCTAAAGAGGTGAACATAGATCAC439                            AspGluTyrIleArgSerGluAlaProLysGluValAsnIleAspHis                               120125130                                                                      GAGACCCGAGAACTGACCAAGACAAACCTACAAGCTGCCACTACCAGT487                            GluThrArgGluLeuThrLysThrAsnLeuGlnAlaAlaThrThrSer                               135140145                                                                      TGCTTCGATGTGGCTCAGGGGAAGACCCGCACATTGATGGAGAAGGAC535                            CysPheAspValAlaGlnGlyLysThrArgThrLeuMetGluLysAsp                               150155160165                                                                   TCCTATCCGCGCTTCCTCAAGTCACCAGCTTATCGCGACCTGGCTGCC583                            SerTyrProArgPheLeuLysSerProAlaTyrArgAspLeuAlaAla                               170175180                                                                      CAAGCCTCGGCCACTTCCACCTCTGCACCCAGCGGCAGCCCAGCTGAG631                            GlnAlaSerAlaThrSerThrSerAlaProSerGlySerProAlaGlu                               185190195                                                                      CCTTCACACACTTGAGCCTCCGCAGCAACCTAGAAGCCATCGGGAAGAGAGGTGG686                     ProSerHisThr*                                                                  200                                                                            AGTCGCCCGTCCCTAAAGCAGCTGCCCTGTGTGGGAGGCAGATCCTGTGCAGCAAGTGCA746                AGAGGATAGTGGAAGGACAGACGGACATCCATCCCCGCAGCTTGAGTGCGAAGAACCCCC806                TCTCCTCCAGATACTGTGGTGGGCCAGTGTAGGAGAGACTCCTCATTTCCAGGACCTGTG866                ACTGAGGGCTGACGACAAGGCTGCGCCGGGTGCTCTGGGGGGAAGGGTGGCCCAGAACTT926                ATACTTTTTACCAGGGCACACACAAAGGGATGCTGGGTGGTTGGGAACCATGAGAACAGG986                AGCCAGAGAGCAGTTATTTAAGGGCCAGACAGTCGGCTTTGGTCCCCGTCTTGATTTCCT1046               CACTCCTGGACTGGGCCTAGAAAAGGCTGTGTGTGTGGAACCTTCATTTCCTGCCCCTTG1106               CCTTCCCAGGGACACCCAGGGCCCCCGAGCTGAGGCTTCTTAATATTCCTGTGCTCATTC1166               TTGCCGTCTCACAGAGGTCAATGAGTCTGTCTGATTCTTGGCCCAGATGAGATTTCTATA1226               CCTCAAAAAACCGGCCTGTGAGCCCCTTTCCGGGTCAATGATGAATCCTGCAAAAGAAGC1286               CATTCTGCTCATGGGACCCTAAGCTGGGTGCTCTCCATGGCCGTCTTGTAGGTGGCCCCC1346               TGCCTTCACCTTTGCAGCAGGGTCTGCTTGTGAACAGGGCTAACTGAGAAGTCTTACTGG1406               GCCCTGCGCTCCTTGGAAGATGGGGTCTAAGGAGGGAGGTGGGAGGAAGGAGACTTCCGG1466               CACAGGGGCCAGCACTCATGTCCAGCAGGTGAGCTAAGGGAGAGAAGTCATTGAAAAATC1526               CATCTAGGAGCCCAAAAGACTAGCTGAAGTCAAACCAGTTCTCTTTGTGGGTATGAAAAT1586               GGGGAAAAGTGTCTCCTACCCCTCACCCTGGTGGAAGGGGAGCCTTGTGCTTCTTTCCTG1646               TTGACATGAGATGCCCACTGTAGTTAGGGGAGAAGCCCGTCAAGGACCAAGAAAGCTCTG1706               GAACAGAAGTTAGCTCAGCCAAGGGAGTATTGCAGATGTGCGGGGAGGGCTGCCTGGAAG1766               GATGGGGCCGGGGGAAACCAACTTGCCTGCCTATCACTTCTGAGTCTTACCGAACAAACT1826               TTCCAAGTTGGGACTTCCAGCACCAGCCACCGAGACGGAGACCTAGGTTGTTTCCCTCTG1886               CACTTGAGGTTTCTCCGGGAGAGAACTCTTTTAAGTATAATATTGTGTTCTGTTGTGTTG1946               TGCCGATTGTCTCGCTGCTATTGTTATTTATTGTGGTTTGTTTGCCTGTACTGAAGAGCC2006               TCAGCTGGAGCTGCTGCCTGATCACGCCTCCTCCCTACCAGACTCTACCTCTGCAAGCCT2066               TGGGAATCACTGAGGGCTGGGGGGGGGGGGGAACGGGACACGGGACTCCCCACTGTGTTA2126               ATATTTATTTATTGTTAACAAAGGGAGCTGGGTTCCTTTATCAGCAGTGTATGTGATCAC2186               TGTTTTTCTGTTTGAGCATGTTATATTCTTGTAAAAAAACCTGAAAATAAAACTCAAAAA2246               TAAAAAAAAAAAAAAAGGGCGGCCGC2272                                                 (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 201 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetCysArgThrLeuAlaThrPheProAsnThrCysLeuGluArgAla                               151015                                                                         LysGluPheLysThrArgLeuGlyIlePheLeuHisLysSerGluLeu                               202530                                                                         SerSerAspThrGlyGlyIleSerLysPheGluTrpAlaSerLysHis                               354045                                                                         AsnLysGluArgSerPheSerGluAspValLeuGlyTrpArgGluSer                               505560                                                                         PheAspLeuLeuLeuAsnSerLysAsnGlyValAlaAlaPheHisAla                               65707580                                                                       PheLeuLysThrGluPheSerGluGluAlaLeuGluPheTrpLeuAla                               859095                                                                         CysGluGluPheLysLysIleArgSerAlaThrLysLeuAlaSerArg                               100105110                                                                      AlaHisHisIlePheAspGluTyrIleArgSerGluAlaProLysGlu                               115120125                                                                      ValAsnIleAspHisGluThrArgGluLeuThrLysThrAsnLeuGln                               130135140                                                                      AlaAlaThrThrSerCysPheAspValAlaGlnGlyLysThrArgThr                               145150155160                                                                   LeuMetGluLysAspSerTyrProArgPheLeuLysSerProAlaTyr                               165170175                                                                      ArgAspLeuAlaAlaGlnAlaSerAlaThrSerThrSerAlaProSer                               180185190                                                                      GlySerProAlaGluProSerHisThr                                                    195200                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATTAACCCTCACTAAATGCTGGGTG25                                                    (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        CATTATGCTGAGTGATATCTTTTTTTTTAA30                                               (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AGGCAAGGGGCAGGAAATGAAG22                                                       (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        ACTAAATGCTGGGTGGTTGG20                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 44 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        AGCGGATCCAAAAAAATGACCATGATTACGCCAAGCTCTAATAC44                                 (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        AGCAATCGATGGAGGCTCAAGTGTGTGAAGGCTCAG36                                         __________________________________________________________________________ 

What is claimed is:
 1. An isolated nucleic acid molecule comprising:(a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of FIGS. 1A-1C (SEQ ID NO:2); (b) the complement of the nucleotide sequence of (a); or (c) a RATH gene or a complement of a RATH gene as contained in ATCC Deposit No.
 98116. 2. An isolated nucleic acid molecule comprising the nucleotide sequence of FIGS. 1A-1C (SEQ ID NO:1).
 3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is cDNA.
 4. An isolated nucleic acid molecule of claim 1, which is RNA.
 5. The isolated nucleic acid molecule of claim 2, further comprising a label.
 6. A delivery complex comprising a nucleic acid molecule of claim 2 and a targeting means.
 7. The delivery complex of claim 6, wherein the targeting means is selected from the group consisting of: a sterol, lipid, virus and a target cell-specific binding agent.
 8. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 missing amino acid residues 60 to 179 of FIGS. 1A-1C (SEQ ID NO:2).
 9. A nucleic acid molecule comprising:(a) a nucleotide sequence encoding a polypeptide comprising amino acid residues 60 to 179 of FIGS. 1A-1C (SEQ ID NO:2); or (b) the complement of the nucleotide sequence of (a).
 10. A recombinant vector containing the nucleotide sequence of claim 1, 2, 3 or
 4. 11. An expression vector containing the nucleotide sequence of claim 1, 2, 3 or 4 operatively associated with a regulatory sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell.
 12. A delivery complex comprising an expression vector of claim 11 and a targeting means.
 13. The delivery complex of claim 12, wherein the targeting means is selected from the group consisting of: a sterol, lipid, virus and a target cell specific binding agent.
 14. A genetically engineered host cell containing the nucleotide sequence of claim 1, 2, 3 or
 4. 15. A genetically engineered host cell containing the nucleotide sequence of claim 1, 2, 3 or 4 operatively associated with a regulatory nucleotide sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in the host cell.
 16. The genetically engineered host cell of claim 15 in which the host cell is prokaryotic.
 17. The genetically engineered host cell of claim 15 in which the host cell is eukaryotic.
 18. A method of making a RATH polypeptide comprising the steps of:(a) culturing the cell of claim 16 in an appropriate culture medium to produce RATH polypeptide; and (b) isolating the RATH polypeptide.
 19. A method of making a RATH polypeptide comprising the steps of:(a) culturing the cell of claim 17 in an appropriate culture medium to produce RATH polypeptide; and (b) isolating the RATH polypeptide. 